We have continued to approach analysis of the rod cyclic nucleotide system with the strategy of component purification and reconstitution of a functional system. We have found that a GTP binding protein is essential for the light activation of rod PDE, and that an additional heat labile macromolecule (called "helper") is necessary for light activated GTPase activity but not for light and GTP dependent activation of PDE. We have identified at least 2 additional loci at which the light activated PDE system appears to be regulated. At the present time the purifications of the GTP binding protein and of the "helper" protein are virtually complete. We are studying the biochemical interactions of the components of the rod system. We are using rhodopsin populations modified by selective proteolysis, phosphorylation or reaction of sulphydral groups, to see what effects these changes will produce on the interaction of rhodopsin with subsequent elements of the cascade. We are also examining the possibility that the peripheral components of amphibian rods would be interchangeable with their counterparts in mammalian rods. Since we have identified additional regulatory components, our reconstitution experiments have to be viewed as partial; i.e., we have been able to reassemble functional subsets of the complete system. Our ultimate goals remain complete characterization and reconstitution of the system.